Author Manuscript NIH Public Access Marie-France Martin ...neuroscience.jhu.edu/files2/publications_Bosmans_F...Deconstructing voltage sensor function and pharmacology in sodium channels

Deconstructing voltage sensor function and pharmacology insodium channels

Frank Bosmans1,2, Marie-France Martin-Eauclaire3, and Kenton J. Swartz11Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, NationalInstitute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892USA2Laboratory of Toxicology, University of Leuven, 3000 Leuven, Belgium3CNRS UMR 6132, CRN2M, Institut Jean Roche, Université de la Méditerranée, Marseille Cedex20, France

AbstractVoltage-activated sodium (Nav) channels are crucial for the generation and propagation of nerveimpulses, and as such are amongst the most widely targeted ion channels by toxins and drugs. Thefour voltage sensors in Nav channels have distinct amino acid sequences, raising fundamentalquestions about their relative contributions to the function and pharmacology of the channel. Herewe use four-fold symmetric voltage-activated potassium (Kv) channels as reporters to examine thecontributions of individual Nav channel S3b-S4 paddle motifs to the kinetics of voltage sensoractivation and to forming toxin receptors. Our results uncover binding sites for toxins fromtarantula and scorpion venom on each of the four paddle motifs in Nav channels and reveal howpaddle-specific interactions can be used to reshape Nav channel activity. One paddle motif isunique in that it slows voltage sensor activation and toxins selectively targeting this motif impedeNav channel inactivation. This reporter approach and the principles that emerge will be useful indeveloping new drugs for treating pain and Nav channelopathies.

Nav channels in nerve and muscle cells open and close, or gate, in response to changes inmembrane voltage1. Mutations in Nav channels can cause a variety of inherited disorders,such as epilepsy and myotonia2, 3. Furthermore, Nav channels are strategically positionedwithin nociceptive signaling pathways, with mutations leading to severe pain disorders4, 5.Although the development of drugs interacting with Nav channels is of widespread medicalimportance6, progress has been slow because the architecture and gating mechanisms ofthese ion channels are complex. Their channel-forming α-subunits contain four homologousdomains (I-IV), or pseudosubunits, each containing six transmembrane segments (S1-S6)(Fig.1a). The S5-S6 segments collectively form a central pore for Na+, with the S1-S4segments from each domain forming the surrounding voltage sensors1. Each of the fourvoltage sensors activate in response to changes in voltage, however, those in domains I-IIIare most important for channel opening, while the one in domain IV is crucial for fastinactivation7-10. Thus, drugs and toxins can interact with multiple regions of Nav channelsto influence their activity, with the four voltage sensors representing rich, yet complextargets. The similarity of the four voltage sensors raises the possibility of multiple binding

Correspondence and requests for materials should be addressed to K.J.S. ([email protected]). .Author Information Reprints and permissions information is available at npg.nature.com/reprints. The authors declare no competingfinancial interests.Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.

NIH Public AccessAuthor ManuscriptNature. Author manuscript; available in PMC 2009 May 13.

Published in final edited form as:Nature. 2008 November 13; 456(7219): 202–208. doi:10.1038/nature07473.

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sites, with occupancy of each having distinct effects on gating. Indeed, seven differentreceptor sites have been proposed for drugs and toxins that interact with Nav channels1, 11,but only a few have been molecularly defined. Here we develop an approach for studyingthe unique contributions of the four voltage sensors in Nav channels to gating andpharmacology. We identify S3b-S4 paddle motifs that can be transplanted from Navchannels into Kv channels without disrupting function, while transferring sensitivity toclassical Nav channel toxins. Our results uncover rich interactions between toxins andmultiple Nav channel paddle motifs, and reveal how domain specific interactions can beused to reshape Nav channel activity.

Transferring paddle motifs between Nav and Kv channelsStudies on voltage sensors in Kv channels have identified an S3b-S4 helix-turn-helix motif,the voltage-sensor paddle, which moves at the protein-lipid interface to drive activation ofthe voltage sensors and opening of the pore12-16. The paddle motif is an importantpharmacological target in Kv channels, as tarantula toxins that partition into membranesinteract with this region to inhibit channel opening12, 17-24. Our initial goal was to determinewhether paddle motifs can be defined in Nav channels and whether they fulfill similarfunctions. Since paddle motifs are portable modules that are interchangeable between Kvchannels and voltage-sensing proteins12, we wondered whether distinct paddle motifs ineach of the four voltage sensors of Nav channels could be transplanted into ahomotetrameric Kv channel to study them in isolation (Fig. 1a). We initially transplanted thepaddle motifs from rNav1.2a and rNav1.4 into the Kv2.1 channel. Although the sequencesof these Nav and Kv channel paddle motifs vary substantially, constructs containing specificS3b-S4 regions of Nav channels result in fully functional channels that display robustvoltage-activated potassium currents (Fig.1b,c, Supplementary Fig.1,2a and SupplementaryTable 1). Functional chimaeras were also obtained by transplanting the paddle motif fromrNav1.4 into Kv1.3 and Shaker Kv channels (Supplementary Fig.1,2b,c, SupplementaryTable 2), demonstrating that paddle motifs of Nav and Kv channels are generallyinterchangeable.

Transferring toxin sensitivity between Nav and Kv channelsWe next asked whether transplanting isolated paddle motifs from Nav channels into Kvchannels faithfully transfers sensitivity to toxins, in which case the Kv channel could beused as a reporter for investigating interactions between toxins and individual Nav channelpaddle motifs. Tarantula toxins related to those targeting paddle motifs in Kv channels12,17-22, 24 (Supplementary Fig.1d) inhibit Nav channels by modifying gating25-27, however,their presumed receptors within the four voltage sensors of Nav channels have yet to bedefined. We focused on three tarantula toxins, PaurTx3, ProTx-I and ProTx-II25-27 andtested whether they interact with the Kv2.1 channels containing each of the four paddlemotifs from rNav1.2a. Remarkably, sensitivity to these toxins can be transferred to Kv2.1along with specific Nav channel paddle motifs (Fig.2a). PaurTx3 has the simplest profile ofthe three, as this toxin only inhibits the Kv channel containing the paddle motif from domainII (Fig.2a, Supplementary Fig.3a and Supplementary Table 3). In contrast, ProTx-I andProTx-II interact with multiple paddle motifs from rNav1.2a. ProTx-I binds to Kv2.1containing the paddle motifs from domain II or IV, whereas ProTx-II interacts with thosefrom domains I, II or IV. (ProTx-I also inhibits Kv2.126, however, the lack of toxinsensitivity for the domain I and III chimaeras support interaction of the toxin with paddlemotifs.) Although it is not straightforward to directly correlate the apparent affinities of Kvchannel paddle chimaeras and Nav channels because the paddles are identical in Kv

Supplementary Information is linked to the online version of the paper at www.nature.com/nature.

Bosmans et al. Page 2

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channels and different in Nav channels, the concentration-dependence for toxin inhibition ofthe two channel types are remarkably similar (Supplementary Fig.4a,b; SupplementaryTable 3). These results show that tarantula toxins often interact with multiple paddle motifsin Nav channels, a scenario that would be difficult to detect using conventionalapproaches27, 28.

Landmark studies on scorpion venom have established the presence of two classes of toxins,the α- and β-scorpion toxins, that interact with the voltage sensors in domains IV and II ofNav channels, respectively1, 29-33. In the conventional view, these toxins interact withsolvent-exposed extracellular loops between S3 and S4 to stabilize the voltage sensors inspecific states8, 31, 34. Whether these toxins interact with the larger S3b-S4 paddle motif, orcan target voltage sensors in domains other than II and IV, is unknown. To explore thesepossibilities we tested whether we could make Kv2.1 sensitive to the classical α- and β-scorpion toxins. As in the case of tarantula toxins, transfer of Nav channel paddle motifs intoKv2.1 renders the channel sensitive to the α-scorpion toxin AaHII, however, in this instanceonly the paddle motif from domain IV can transfer toxin sensitivity (Fig.2a; SupplementaryFig.3a). Although the apparent affinity of AaHII for the domain IV chimaera is considerablyless than for rNav1.2a (Supplementary Fig.4d; Supplementary Table 3), mutations within S4can increase the apparent affinity by over 10-fold (Fig.3d,4c; Supplementary Fig.4d). Theseresults suggest that α-scorpion toxins interact with the paddle motif from domain IV, but themodest affinity of the paddle chimaera leaves open the possibility that other regions of Navchannels contribute to the toxin receptor35.

We also examined whether sensitivity to the β-scorpion toxin TsVII could be transferredwith Nav channel paddle motifs. In contrast to AaHII, transfer of the paddle motifs fromdomains II, III or IV from rNav1.2a renders the Kv channel sensitive to TsVII (Fig.2a;Supplementary Fig.3a), revealing that β-scorpion toxins can interact with multiple Navchannel paddle motifs. This result is surprising because studies on rNav1.4 suggest thatTsVII only binds to the voltage sensor in domain II36, 37. To explore whether scorpion toxininteractions vary between Nav channel subtypes, we tested the sensitivity of the rNav1.4paddle chimaeras to TsVII. In contrast to what is observed with the rNav1.2a paddles, TsVIIcan interact selectively with the domain II paddle from rNav1.4 (Fig.2b; Supplementary Fig.3b). Although the apparent affinities of TsVII for the rNav1.2a paddle chimaeras aresignificantly lower than the rNav1.2a channel, the rNav1.4 domain II paddle chimaera iscomparable (Supplementary Fig.4c, Supplementary Table 3). An interesting feature of TsVIIis that the β-scorpion toxin stabilizes a closed state in the paddle chimaeras (Fig.2), whereasthe toxin stabilizes the open state of rNav1.2a (Fig.4b) and rNav1.437 channels. β-scorpiontoxins can also stabilize a closed state in certain Nav channels32, indicating that open statestabilization is not always observed. One explanation is that toxin binding to the paddleintrinsically stabilizes a closed state, but in certain Nav channels the toxin can stabilize anopen state when it also interact with other regions32, 33. Taken together, these data suggestthat both tarantula and scorpion toxins interact with paddle motifs, that multiple paddlemotifs can be targeted by individual toxins, and that these interactions can differ betweenNav channel subtypes.

Critical residues in the toxin-paddle interactionTo further explore the extent to which toxins interact with S3b-S4 paddle motifs, and toidentify mutants for testing whether these toxin-paddle interactions actually occur in Navchannels, we mutated each residue in the transferred paddle regions and measured changesin toxin affinity (Fig.3; Supplementary Fig.4 and Supplementary Tables 4-7). One strikingfeature of the data is that mutations distributed throughout the S3b-S4 paddle motifs causedramatic perturbations in the apparent affinity for both tarantula and scorpion toxins,



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suggesting that these toxins target the larger paddle motif. Many of the most influentialmutations are of hydrophobic residues, but mutations of polar residues such as Glu residuesin S3b and Arg residues in S4 also have pronounced effects. The present mutagenesis of thedomain IV paddle motif correctly identify several mutants previously shown to weaken α-scorpion toxin affinity in Nav channels31, but reveal a larger number of influentialmutations, many of which have more pronounced effects on toxin affinity. This highlightsan advantage of the reporter approach, which allows individual toxin-paddle interactions tobe studied in isolation; toxins can interact with up to four different paddle motifs in Navchannels and the effects of mutations in any one of those can be quite subtle because theothers remain unaltered.

Another striking feature of these paddle scans is that mutations perturbing toxin-paddleinteractions are rather unique for each toxin-paddle pair. For example, both ProTx-I andProTx-II interact with the paddle motif from domain II, however, the mutants that weakentoxin affinity only partially overlap. In addition, the mutagenesis results for ProTx-IIinteracting with paddle motifs from domains I, II and IV identify many influential mutationsthat differ between the three paddles. This comparison suggests that toxin-paddleinteractions do not obey a general lock- and-key mechanism, but that the interfaces varyconsiderably.

Validating toxin-paddle interactions in rNav1.2aTo validate that the toxin-paddle interactions identified here actually occur in Nav channels,we reconstituted the most influential paddle mutants into rNav1.2a and measured changes intoxin affinity. For tarantula toxins, we examined mutations influencing ProTx-II affinitysince this toxin interacts with three of the four Nav channel paddle motifs. Mutations withinthe paddle motifs of domains I, II and IV weaken the interaction of ProTx-II with rNav1.2(Fig.4a), suggesting that the toxin interacts with all three paddle motifs in the Nav channel.The mutations tend to have smaller effects in the Nav channel when compared to the paddlechimaeras (compare Fig.4a with Fig.3a,b,d), which makes sense because the mutations inthe Nav channel alter only one of three targeted paddle motifs, whereas in the Kv channelchimaera they alter all four. For the β-scorpion toxin TsVII, we examined mutations withinthe paddle motif of domain III and found that they diminish the hyperpolarizing shiftproduced by moderate toxin concentrations (Fig.4b), confirming that the toxin interacts withdomain III (in addition to its canonical interaction with domain II). Finally, in the case of theα-scorpion toxin AaHII, we reconstituted a gain-of-function mutation into rNav1.2a andobserved that the mutation also increases the apparent affinity of the toxin in the Navchannel (Fig.4c). These results demonstrate that the toxin-paddle interactions uncoveredusing the reporter approach actually occur in Nav channels.

Unique character of the domain IV paddle motifThe differential coupling of the four voltage sensors in Nav channels to opening andinactivation8, 10, 38-40 is accompanied by differences in the kinetics of voltage sensoractivation, with the voltage sensor of domain IV moving slower than the other three10.Given that the paddle motif resides in a relatively unconstrained environment in contact withthe surrounding lipid membrane, and that it is the region of the voltage sensor that flexes inresponse to changes in voltage12-16, 41, we wondered whether the paddle motif itself mightbe responsible for these kinetic differences. To explore this possibility we measured thekinetics of activation (opening) and deactivation (closing) of the four rNav1.2a paddlechimaeras in Kv2.1 in response to membrane depolarization and repolarization, respectively(Fig.5). At most voltages, the time constants obtained by fitting single exponential functionsto current relaxations are notably slower for the domain IV paddle chimaera compared to the



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other chimaeras and to Kv2.1. This difference is not unique to chimaeras between therNav1.2a and Kv2.1 channel, but is even more pronounced when transplanting paddle motifsfrom rNav1.4 into Kv2.1, Kv1.3 or Shaker Kv channels (Supplementary Fig.2). Theseresults demonstrate that the paddle motif in domain IV of Nav channels determines theslower movement of this voltage sensor in response to changes in membrane voltage.

Shaping Nav channel activity with domain-selective interactionsA particularly intriguing outcome of looking at toxin-paddle interactions in isolation is thatpatterns emerge between the domains targeted and the effects of the toxin on Nav channelactivity. Inspection of the profiles for toxin-paddle interactions (Fig.2) reveals that bytargeting paddle motifs in domains I, II or III, the overall effect of a toxin is to influenceopening of Nav channels, irrespective of whether domain IV is also targeted. The tarantulatoxins PaurTx3, ProTx-I, ProTx-II, as well as the β-scorpion toxin TsVII, are examples ofthis relationship. In contrast, the requirements for influencing inactivation are morestringent; to do so a toxin needs to selectively target the paddle motif in domain IV, as is thecase with the α-scorpion toxin AaHII. To test whether this pattern is generally applicableacross different toxin families, we searched for a tarantula toxin that selectively interactswith the paddle motif in domain IV and asked whether it influences inactivation. Using ourreporter constructs, we screened various tarantula toxins, including VSTx142, GxTx-1E43,HaTx18 and SGTx144, all of which interact with paddle motifs in Kv channels12, 17-21, 24, 44

and are related in sequence to the Nav channel toxins already studied (Supplementary Fig.1d). VSTx1 (10 μM) and GxTx-1E (500 nM) are inactive against the Nav paddle chimaeras(not shown), whereas both HaTx and SGTx1 exhibit robust effects (Fig.6a). HaTx interactswith paddle motifs from domains I, II and IV, similar to ProTx-II, whereas SGTx1 interactsselectively with the domain IV paddle chimaera, the profile we sought to find. When testedagainst rNav1.2a, HaTx exhibits robust inhibitory effects, similar to ProTx-II, withoutsignificantly altering inactivation (Fig.6b). In contrast, SGTx1 does not inhibit rNav1.2a oralter channel opening, but dramatically reduces the extent of inactivation (Fig.6c), behavingas an α-scorpion toxin. These results suggest that for a toxin to influence inactivation, itmust selectively interact with the paddle motif in domain IV; any additional interactionswith the other paddles will alter channel opening.

DiscussionOur results with isolating Nav channel paddle motifs using Kv channels as reporters havethree fundamental implications for the function and pharmacology of Nav channels. First,they reveal that paddle motifs can determine the kinetics of voltage sensor activation. ForNav channels to function properly, inactivation must proceed more slowly than opening, aproperty that has been attributed to slower activation of the voltage sensor in domain IV1, 8,10, 38, 45. Our results show that the paddle motif in domain IV is unique because itsystematically slows activation of any voltage sensor into which it is incorporated (Fig.5;Supplementary Fig.2). The paddle motif resides in a relatively flexible environment andmoves in contact with the surrounding lipid membrane12-16, 41, raising the intriguingpossibility that interactions of the domain IV paddle motif with lipids are unique and areresponsible for slowing voltage sensor activation. Other aspects of Nav channel function areunlikely to be determined by the paddle motif (e.g. cooperative voltage sensor activation10,37, 46), but the approaches described here may help to identify them.

Second, our results reveal rich interactions of tarantula and scorpion toxins with paddlemotifs and establish a number of principles for toxin-paddle interactions in Nav channelswhere the four paddle motifs are not equivalent. Although α- and β-scorpion toxins areknown to interact with domains IV and II, respectively31, 32, the present results demonstrate



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that each of the paddle motifs are targeted by toxins (Fig.2). In fact, multiple paddle motifsare often targeted by a toxin. Even the β-scorpion toxins can interact with other domains inaddition to the canonical interaction with domain II. One unexpected finding is that theprofiles of toxin-paddle interactions can differ between subtypes of Nav channels,suggesting that such profiles may be a way of distinguishing Nav channels. Our mutagenesisresults with the four paddle motifs in rNav1.2a also show that the pattern of mutants alteringtoxin affinity is specific for each toxin-paddle pair (Fig.3). The most influential mutants canvary considerably for one toxin interacting with different paddles, or for two related toxinsinteracting with a single paddle motif.

Finally, the profiles of toxin-paddle interactions that emerge from our studies reveal animportant relationship between the effect of a toxin on Nav channel activity and domain-specific interactions. Alterations in channel opening can be achieved with molecules thatinteract with any of the first three paddle motifs in Nav channels without regard for whetherdomain IV is also targeted (e.g. HaTx, ProTx-II, TsVII), whereas influencing inactivationrequires that a molecule interact exclusively with the paddle in domain IV (e.g. SGTX1,AaHII). These domain-specific interactions have important implications for designing drugsto reshape Nav channel activity. Channelopathies like idiopathic ventricular fibrillation47

and Long QT syndrome type-348 are associated with accelerated Nav channel inactivation,which could be restored by selectively targeting the domain IV paddle motif. In contrast, thehyperpolarization of Nav channel opening seen in disorders like generalized epilepsy withfebrile seizures type 249 and hyperkalaemic periodic paralysis50 could be managed withdrugs targeting any paddle motif within the first three domains. Our reporter approach foridentifying toxins that specifically target the paddle motif in domain IV (Fig.6) provides aconceptual example for identifying drugs that interact with specific paddle motifs in Navchannels.

METHODS SUMMARYChannel constructs were expressed in Xenopus oocytes21 and studied using two-electrodevoltage-clamp recording techniques. For Kv channel experiments, the external recordingsolution contained: 50mM KCl, 50mM NaCl, 5mM HEPES, 1mM MgCl2 and 0.3mMCaCl2, pH 7.6 with NaOH. KCl was replaced by RbCl for Shaker experiments. For Navchannel experiments, the external recording solution contained 96mM NaCl, 2mM KCl,5mM HEPES, 1mM MgCl2 and 1.8mM CaCl2, pH 7.6 with NaOH.

Voltage-activation relationships were obtained by measuring tail currents for Kv channels orsteady-state currents and calculating conductance for Nav channels, and a single Boltzmannfunction was fitted to the data. Occupancy of closed or resting channels by toxins wasexamined using negative holding voltages where open probability was low, and the fractionof unbound channels (Fu) was estimated using depolarizations that are too weak to opentoxin-bound channels, as described previously17-21, 24, 44. The apparent equilibriumdissociation constant (Kd) for toxin interaction with Kv channel constructs was calculatedassuming four independent toxin-binding sites per channel, with single occupancy beingsufficient to inhibit opening in response to weak depolarizations (See Full Methods,Supplementary Fig. 4, 5c and Supplementary Table 3 for information on Kd measurements.).

AcknowledgmentsWe thank J.W. Kyle, D.A. Hanck and A.L. Goldin for the rNav1.2a, rNav1.4 and β1 clones, C. Deutsch for Kv1.3,M.M. Smith for GxTx-1E, K.M. Blumenthal and J.B. Herrington for ProTx-II, L.D. Possani for a sample of Ts1 (orTsVII), the NINDS DNA sequencing facility for DNA sequencing, and the NINDS protein sequencing facility formass spectrometry and peptide sequencing. We thank AbdulRasheed A. Alabi for helping with Kv and Nav channelalignments and Tsg-Hui Chang for assistance with Nav channel mutants. We also thank AbdulRasheed A. Alabi,



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Miguel Holmgren, Mark Mayer, Mirela Milescu, Joe Mindell, Andrew Plested, Shai Silberberg and members of theSwartz laboratory for helpful discussions. This work was supported by the Intramural Research Program of theNINDS, NIH and by a NIH-FWO postdoctoral fellowship to F.B.

Appendix

FULL METHODSChannel and chimaera constructs

Chimaeras and point mutations were generated using sequential polymerase chain reactions(PCR) with Kv2.1Δ719, 51, Kv1.352, Shaker53, rNav1.2a (neuronal type) 54 or rNav1.4(muscle type)55 as templates. The Kv2.1Δ7 construct contains seven point mutations in theouter vestibule19, rendering the channel sensitive to agitoxin-2, a pore-blocking toxin fromscorpion venom56. The Shaker construct contains a deletion of residues 6 to 46 to removeinactivation57. The DNA sequence of all constructs and mutants was confirmed byautomated DNA sequencing and cRNA was synthesized using T7/SP6 polymerase(mMessage mMachine kit, Ambion) after linearizing the DNA with appropriate restrictionenzymes.

Spider and scorpion toxin purificationPaurTx325 was purified from the venom of Phrixotrichus auratus (Spider Pharm) using anew two-step HPLC protocol (Supplementary Fig.5a). Identity and purity were determinedwith mass spectrometry and automated peptide sequencing. Hanatoxin was purified fromGrammostola spatulata venom (Spider Pharm) as described previously58. SGTx1 andVSTx1 were synthesized using solid-phase chemical methods, folded in vitro and purified asdescribed previously44, 59. AaHII from Androctonus australis hector venom (animalscollected near Tozeur, Tunisia), and TsVII from Tityus serrulatus venom (generous giftfrom Prof. C. Diniz) were purified as described previously60, 61. Synthetic ProTx-I wasacquired from Peptides International (USA). ProTx-II was generously provided by K.M.Blumenthal (SUNY, Buffalo) and J.B. Herrington (Merck Research Labs, Rahway, NJ).GxTx-1E was generously provided by M.M. Smith (Merck Research Labs, Rahway, NJ).The plant alkaloid veratridine (VTD; Sigma, USA) is used as a negative control in Fig.2since the binding site of this lipid-soluble compound is thought to consist of the S6transmembrane segments62. Toxins were kept at -20 °C. Before experiments, toxin aliquotswere dissolved in appropriate solutions containing 0.1% BSA (or 1% BSA in the case ofTsVII).

Two-electrode voltage-clamp recording from Xenopus oocytesChannel constructs were expressed in Xenopus oocytes21 and studied following 1-2 daysincubation after cRNA injection (incubated at 17 °C in 96mM NaCl, 2mM KCl, 5mMHEPES, 1mM MgCl2 and 1.8mM CaCl2, 50μg/ml gentamycin, pH 7.6 with NaOH) usingtwo-electrode voltage-clamp recording techniques (OC-725C, Warner Instruments) with a150μl recording chamber. Data were filtered at 1 kHz and digitized at 10 kHz using pClampsoftware (Axon). Microelectrode resistances were 0.1-1MΩ when filled with 3M KCl. Formost Kv channel experiments, the external recording solution contained: 50mM KCl, 50mMNaCl, 5mM HEPES, 1mM MgCl2 and 0.3mM CaCl2, pH 7.6 with NaOH. KCl was replacedby RbCl for Shaker experiments. For Nav channel experiments, the external recordingsolution contained 96mM NaCl, 2mM KCl, 5mM HEPES, 1mM MgCl2 and 1.8mM CaCl2,pH 7.6 with NaOH. For Nav channel experiments, oocytes were co-injected with the β1subunit in a 1:5 molar ratio. All experiments were performed at room temperature (∼22 °C).Leak and background conductances, identified by blocking the channel with agitoxin-2,



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have been subtracted for all of the Kv channel currents shown21. Tetrodotoxin subtraction(TTX) or an online P/-4 prepulse protocol was used to isolate Nav channel currents.

Analysis of channel activity and toxin-channel interactionsVoltage-activation relationships were obtained by measuring tail currents for Kv channels orsteady-state currents and calculating conductance for Nav channels, and a single Boltzmannfunction was fitted to the data according to: I/Imax = (1 + e-zF(V - V1/2)/RT)-1 where I/Imax isthe normalized tail-current amplitude, z is the equivalent charge, V1/2 is the half-activationvoltage, F is Faraday’s constant, R is the gas constant and T is temperature in Kelvin.

Occupancy of closed or resting channels by toxins was examined using negative holdingvoltages where open probability was low, and the fraction of unbound channels (Fu) wasestimated using depolarizations that are too weak to open toxin-bound channels, asdescribed previously17-21, 24, 44. After addition of the toxin to the recording chamber, theequilibration between the toxin and the channel was monitored using weak depolarizationselicited at 5-10s intervals. For all channels, we recorded voltage-activation relationships inthe absence and presence of different concentrations of toxin. The ratio of currents (I/I0)recorded in the presence (I) and absence (I0) of toxin was calculated for various strengthdepolarizations, typically -70 mV to +10 mV. The value of I/I0 measured in the plateauphase at voltages where toxin-bound channels do not open was taken as Fu (SupplementaryFig.5b,c). The apparent equilibrium dissociation constant (Kd) for Kv channels wascalculated according to Kd = ((1/(1 - Fu1/4)) - 1)[Toxin] assuming four independent toxin-binding sites per channel, with single occupancy being sufficient to inhibit opening inresponse to weak depolarizations. For all chimaeras and mutants, voltage protocols wereadjusted appropriately so that the plateau phase in the I/I0-voltage relationship was welldefined. Example traces showing the inhibitory activity of tarantula toxins were taken forrelatively weak depolarizations within the plateau phase for that particular channelconstruct. Off-line data analysis was performed using Clampfit (Axon), Origin 7.5(Originlab) and Microsoft Solver (Microsoft Excel).

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Figure 1. Transfer of the voltage sensor paddle motifs from rNav1.2a to Kv2.1a, Cartoon depicting the paddle motif transfer from the Nav channel S1-S4 voltage sensor ofdomain II into Kv2.1. Purple: domain I paddle (DI), red: domain II paddle (DII), blue:domain III paddle (DIII) and green: domain IV paddle (DIV). Color code will be used in allfigures. b,c, Families of potassium currents (b) and tail current voltage-activationrelationships (c) for each chimaera (n = 18, error bars represent s.e.m.). Holding voltage was-90mV and the tail voltage was -50mV (-80mV for DIII). Scale bars in (b) are 2 μA and 100ms.



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Figure 2. Sensitivity of rNav1.2a paddle chimaeras to extracellular toxinsa, Effects of toxins on Kv2.1 and chimaeras where paddle motifs were transferred fromrNav1.2a into Kv2.1. Normalized tail current voltage-activation relationships are shownwhere tail current amplitude is plotted against test voltage before (black) and in the presenceof toxins (other colors). Data are grouped per toxin (horizontally) and per chimaera or wild-type Kv2.1 (vertically). Concentrations used are 100nM PaurTx3, ProTx-I and ProTx-II;1μM AaHII; 500nM TsVII and 100μM VTD. b, Effects of TsVII (50 nM) on rNav1.4paddle chimaeras. n = 3-5 and error bars represent s.e.m. The plant alkaloid veratridine(VTD) is used as a negative control. The holding voltage was -90mV, test pulse durationwas 300 ms, and the tail voltage was -50mV (-80mV for DIII).



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Figure 3. Scanning mutagenesis of Nav channel paddle motifsa-d, Ala-scan of the separate rNav1.2a paddle motifs in the DI to DIV chimaeras wherechanges in apparent toxin affinity (Kd

mut/Kdcont) are plotted for individual mutants. See Full

Methods, Supplementary Fig. 4, 5c and Supplementary Table 3 for information on Kdmeasurements. Most of the residues within the rNav1.2a paddle in the four chimaeraconstructs were individually mutated to Ala (except for native Ala residues, which weremutated to Val). The dashed line marks a value of 1. Each mutant was initially examinedusing a concentration near the Kd value determined for the control chimaera (seeSupplementary Fig. 4). Mutants with a Kd

mut/Kdcont value greater than five were further

examined using a wider range of concentrations. Glu residues marked with asterisks were



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also mutated to a Lys. Bar diagrams are approximately aligned according to the sequencealignment of the different paddles (see Supplementary Fig. 1). Mutants without acorresponding bar did not result in functional channels. Residues with a grey backgroundwere used in subsequent tests (Fig. 4). Mutation of two underlined residues in (d) results inan increase in AaHII affinity (Kd = 235 ± 24nM for R1629A, 205 ± 23nM for L1630A and1902 ± 102nM for the DIV chimera). n = 3-5 for each toxin concentration and error barsrepresent s.e.m.



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Figure 4. Reconstitution of paddle mutants into rNav1.2a and their effects on toxin-channelinteractionsa, Concentration-dependence for ProTx-II inhibition of rNav1.2a and selected mutantsplotted as fraction unbound (Fu) measured at negative voltages. Solid lines are fits of theHill equation to the data with apparent Kd values shown. See Supplementary Fig. 5b andSupplementary Table 3 for further information. b, Normalized conductance-voltagerelationships for rNav1.2a and two DIII mutants before and after addition of 50nM TsVII.Arrow indicates toxin effect or lack thereof. Holding voltage was -90 mV. c, rNav1.2amutation L1630A increases affinity for AaHII. Sodium currents were elicited by a



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depolarization to -15 mV from a holding voltage of -90 mV. Green trace is after AaHIIaddition.



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Figure 5. Kinetics of opening and closing for rNav1.2a/Kv2.1 chimaerasa, Representative macroscopic currents (black) showing channel activation (left) andchannel deactivation (right) using the following voltage protocols: activation, 10 mVincrementing steps to voltages between -40mV and +100mV from a holding potential of -90mV; deactivation, 10 mV incrementing steps to voltages between 0mV and -100mV(-120mV for DIII) from a test voltage of between +80 and +100 mV (holding potential is-90 mV). Superimposed red curves are single exponential fits to the current records afterinitial lags in current activation. b, Mean time constants (τ) from single exponential fits tochannel activation (filled circles) and deactivation (open circles) plotted as a function of thevoltage at which the current was recorded. n = 4-8 and error bars represent s.e.m.



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Figure 6. Identifying a tarantula toxin selective for the paddle motif in domain IVa, Potassium currents elicited by depolarizations near the foot of the voltage-activation curvefor Kv2.1 and chimaeras in the absence and presence of 50nM HaTx or 100nM SGTx1. Theholding voltage was -90mV, test pulse duration was 300 ms, and the tail voltage was -50mV(-80mV for DIII). b, Conductance-voltage relationships for rNav1.2a before and afteraddition of 50nM HaTx (left), normalized to the maximal conductance in control. Sodiumcurrent elicited by a depolarization to -30 mV before and after addition of 50nM HaTx(right). c, Conductance-voltage relationship of rNav1.2a before and after addition of 100nMSGTx1 (left), individually normalized to the maximal conductance in either control or thepresence of toxin. Sodium current elicited by a depolarization to -15 mV before and after



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addition of 100nM SGTx1 (right). n = 3-5 for each toxin concentration and error barsrepresent s.e.m (b, c).



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Author Manuscript NIH Public Access Marie-France Martin ...neuroscience.jhu.edu/files2/publications_Bosmans_F...Deconstructing voltage sensor function and pharmacology in sodium channels